Ehrlichia chaffeensis infects monocytes and macrophages, and causes a potentially fatal emerging infectious disease called Human monocytic ehrlichiosis (HME). The complete E. chaffeensis Arkansas genome sequence was published in 2006. This is also the only strain for which experimental pathogenesis data are available. Severity of HME varies from asymptomatic infection to death. Our overall hypothesis in the proposed study is that comparative genome sequence analysis combined with comparative pathogenesis studies of multiple E. chaffeensis isolates can provide valuable insights into potential E. chaffeensis virulence determinants and new intervention targets. The specific aims are as follows: 1. Identify polymorphic genes or genomic regions of E. chaffeensis strains by CGH using densely tiled microarray and confirm by DNA sequencing. 2. Determine phenotypes of E. chaffeensis strains in established animal models. 3. Analyze temporal transcriptome profiles of hosts in response to E. chaffeensis strains of distinct virulence, and confirm the results using quantitative RT-PCR, and antibodies specific to selected cytokines and host signaling molecules. 4. Functionally characterize polymorphic E. chaffeensis genes associated with virulence 1) by expressing the virulent and the avirulent versions of recombinant proteins or by transfection of host cells with the candidate virulent gene to study their effects on target host cell functions;2) by making antibodies to the recombinant proteins and localizing the proteins by confocal immunofluorescent or immunogold electron microscopy: 3) by identifying host interacting proteins by immunoprecipitation of host/bacterial protein complexes and by yeast two-hybrid system: 4) by determining in vitro infection neutralizing effects of the antibodies;and/or by immunizing immunocompetent animals with recombinant proteins or passively immunizing SCID mice with specific antibodies, and challenging them with the virulent strain. The proposed study will identify novel E. chaffeensis virulence determinants and their pathogenic mechanisms. The results may point to potential chemotherapy, chemopreventive and/or vaccine candidates for treatment and prevention of human ehrlichiosis.